Quercetin was methylated with two O-methyltransferases (OMTs) expressed in E. coli. A construct (RSOMT) was designed to express two OMTs: ROMT-9, which methylates specifically at the 3'-hydroxyl group of quercetin and SOMT-2, which methylates at the 4'-hydroxyl group. Both OMT genes were driven by T7 promoters and had ribosome binding sites. Both ROMT-9 and SOMT-2 were successfully expressed in E. coli transformant harboring RSOMT. Reaction products of quercetin with E. coli transformant containing RSOMT showed two methylation products that corresponded to the 3'-methylated and the 3',4'-dimethylated quercetin, which were confirmed by NMR. More than 90% of quercetin was converted into the 3',4'-dimethylated quercetin after 24 h incubation with E. coli transformant harboring RSOMT.